Doctor of Philosophy / Department of Anatomy and Physiology / Michael J. Kenney / It is well established that normal aging is associated with progressive increases in efferent sympathetic nerve discharge (SND). Type II diabetes, obesity, heart failure, and hypertension are pathologies that have been attributed to both the processes of aging and sympathetic dysfunction, exemplifying the importance of understanding central regulation of SND during aging. However, the central mechanisms mediating altered SND with advancing age remain unclear. The rostral ventral lateral medulla (RVLM) is a brainstem region critically involved in setting the basal level of sympathetic outflow and cardiovascular function. Indeed, the RVLM is the only presympathetic region that when bilaterally inactivated results in profound reductions in both SND and arterial pressure. Glutamatergic influences in RVLM activity are powerfully inhibited by tonic GABAergic neural inputs originating from the caudal ventral lateral medulla (CVLM); effects that are mediated by GABAA receptors located on presympathetic neuronal cell bodies within the RVLM. In the present study we proposed that reductions in GABA[subscript A] receptor subunit gene expression may reflect withdrawal of GABAergic tone in the RVLM thereby contributing to the basal sympathetic activation that occurs with advancing age. Therefore, the objective of the current study was to identify age-related changes in the constitutive expression of genes related to GABAergic and muscarinic, nicotinic and dopaminergic receptor systems due to their reported involvement in modulating GABA[subscript A] receptor function, in the RVLM of adult young (3-5 mo. old), middle-aged (12 mo. old), weight stable presenescent (24-25 mo. old) and senescent (>24 mo. old) Fischer 344 (F344) rats using a commercially available real-time PCR array. Real-time analysis revealed nonuniform and age-associated changes in the RVLM GABA, muscarinic, nicotinic and dopaminergic neurotransmitter gene expression profile between young and middle-aged F344 rats. Heterogeneous expression of genes related to these neurotransmitters was also observed between presenescent and senescent F344 rats. Our results suggest that potential changes in neurotransmitter synthesis and degradation, uptake, transport, signaling and receptor subunit composition may account for the sympathoexcitatory state that is commonly observed in the aged.

Due to various shortcomings of the visual system, some visual stimuli can only be identified with 100% accuracy if they are shown for a certain amount of time. This time can be measured using the Inspection Time (IT) paradigm. In an IT task, a “pi” figure with differing leg lengths is typically presented briefly (e.g., 20-200 ms) and is then immediately masked to prevent retinal afterimages. Participants are subsequently required to choose which of the two legs was longer. The objective of this task is to determine the shortest amount of time the pi figure needs to be shown for it to be perceived with 80% accuracy. Given that visual processing has been shown to be altered during and /or prior to a movement, the present experiment sought to test how the requirement to perform a motor task affected IT. Twenty-eight participants took part in the experiment, which was comprised of three conditions: no-movement (NM), peak velocity (PV), and foreperiod (FP). In the NM condition, participants grasped a manipulandum and engaged in the IT paradigm. At the end of every trial, participants verbally stated which leg they believed was longest. In the PV condition participants made a rapid movement to a target, and the IT stimulus was presented when their limb reached peak velocity. Finally in the FP condition the IT stimulus was presented during foreperiod (FP). In all three conditions the IT stimulus was randomly presented from between 15-105 ms (in 15 ms increments) and masked for 400 ms. Results showed no significant differences on the IT task between the NM and FP conditions, suggesting no visual upregulation during foreperiod. However, IT performance was significantly poorer in the PV condition in comparison to both the NM and FP condition, suggesting a visual downregulation at that particular movement kinematic.

Projections from the Nucleus Accumbens (NAc) to the Ventral Pallidum (VP) play a critical role in motivation and reward. Rewards and reward-associated cues are thought to alter this pathway by suppressing GABA release to the VP, however, the role of the NAc to VP pathway has never been investigated with regard to nicotine self-administration. We hypothesized that increasing GABAergic signaling from the NAc to the VP would decrease the reinforcing effects of nicotine. To increase GABA release, CRE dependent DREADD was expressed in the NAc of male rats. Administration of low dose clozapine activated the DREADD receptor and showed a reduction in responding for nicotine suggesting that activation of the NAc to VP pathway reduced reinforcement. However, a nonspecific effect was observed leading to future investigation of locomotor activity. Immunohistochemistry and microdialysis were used to confirm DREADD expression in the NAc and increased extracellular GABA in the VP.

Ventrolateral medullary neurons have important roles in cardiorespiratory coordination. A rostral extension of the ventral respiratory column (RVRC), including the retrotrapezoid nucleus (RTN), has neurons responsive to local perturbations of CO2 / pH. Respiratory-modulated firing patterns of RVRC neurons are attributed to influences of more caudal (CVRC) neurons. These circuits remain poorly understood. This study addressed the hypothesis that both local interactions and influences from the CVRC shape rostral neuron discharge patterns and responses. Spike trains from 294 rostral and 490 caudal neurons were recorded with multi-electrode arrays along with phrenic nerve activity in 14 decerebrate, vagotomized cats. Overall, 214 rostral and 398 caudal neurons were respiratory-modulated; 124 and 95, respectively, were cardiac-modulated. Subsets of these neurons were evaluated for responses to sequential, selective, transient stimulation of central and peripheral chemoreceptors and arterial baroreceptors. In 5 experiments, Mayer wave-related oscillations (MWROs) in neuronal firing rates were evoked, enhanced, or reduced following central chemoreceptor stimulation. Overall, 174 of the rostral neurons (59.5%) had short- time scale correlations with other RVRC neurons. Of these, 49 triggered cross-correlograms with RVRC targets yielding 330 offset features indicative of paucisynaptic actions from a total of 2,884 rostral pairs evaluated. Forty-nine of the CVRC neurons (10.0%) were triggers in 142 CVRC-RVRC correlograms - from a total of 8,490 - with offset features indicative of actions on RVRC neurons. Correlation linkage maps support the hypothesis that local circuit mechanisms contribute to the respiratory and cardiac modulation of RVRC neurons and their responses to chemoreceptor and baroreceptor challenges.

Alcohol use disorder is a debilitating disorder affecting nearly 5% of people in the United States. Despite the prevalence of alcohol use disorder few affected individuals seek treatment and of those who do many will relapse. This highlights a need to develop new treatments for alcohol use disorder that are both more accessible and more effective. This dissertation characterizes a novel pathway involved in ethanol enhancement of dopamine levels in the nucleus accumbens as well as investigating alterations in dopamine 2 receptor expression and function following an acute dose of ethanol. This was done by using microdialysis to measure dopamine levels in the nucleus accumbens, single-unit recordings of dopamine neurons in the ventral tegmental area to measure dopamine neuron activity and place conditioning to measure the rewarding properties of the intravenous dopamine and ethanol. It was found that activation of peripheral dopamine 2 receptors by intravenous dopamine enhanced dopamine levels in the nucleus accumbens and dopamine neuron firing rate in the ventral tegmental area. Additionally, intravenous dopamine produced a modest conditioned place preference. Domperidone, a peripheral dopamine 2 receptor antagonist blocked each of these effects. Further, domperidone blocked ethanol enhancement of dopamine release in the nucleus accumbens and bidirectionally modulated the sedating effects of ethanol depending on the dose of ethanol administered. The involvement of peripheral dopamine 2 receptors in ethanol reward could not be ascertained in these studies as domperidone produced a weak conditioned place aversion. Finally, acute ethanol was found to enhance dopamine 2 receptor expression in the nucleus accumbens and medial prefrontal cortex while also enhancing dopamine 2 receptor expression on NK and B cells. Additionally, ethanol was found to reduce desensitization of dopamine 2 receptors in the ventral tegmental area. These results demonstrate that activation of peripheral dopamine 2 receptors can enhance dopamine levels in the nucleus accumbens and that this effect has relevance in understanding the effects of ethanol on dopamine release in the mesolimbic pathway. These results also provide evidence for transient upregulation of dopamine 2 receptors in the brain and on leukocytes suggesting that dopamine 2 receptor levels on leukocytes may be a useful biomarker for central dopamine function.

Alcohol use disorder is a debilitating disorder affecting nearly 5% of people in the United States. Despite the prevalence of alcohol use disorder few affected individuals seek treatment and of those who do many will relapse. This highlights a need to develop new treatments for alcohol use disorder that are both more accessible and more effective. This dissertation characterizes a novel pathway involved in ethanol enhancement of dopamine levels in the nucleus accumbens as well as investigating alterations in dopamine 2 receptor expression and function following an acute dose of ethanol. This was done by using microdialysis to measure dopamine levels in the nucleus accumbens, single-unit recordings of dopamine neurons in the ventral tegmental area to measure dopamine neuron activity and place conditioning to measure the rewarding properties of the intravenous dopamine and ethanol. It was found that activation of peripheral dopamine 2 receptors by intravenous dopamine enhanced dopamine levels in the nucleus accumbens and dopamine neuron firing rate in the ventral tegmental area. Additionally, intravenous dopamine produced a modest conditioned place preference. Domperidone, a peripheral dopamine 2 receptor antagonist blocked each of these effects. Further, domperidone blocked ethanol enhancement of dopamine release in the nucleus accumbens and bidirectionally modulated the sedating effects of ethanol depending on the dose of ethanol administered. The involvement of peripheral dopamine 2 receptors in ethanol reward could not be ascertained in these studies as domperidone produced a weak conditioned place aversion. Finally, acute ethanol was found to enhance dopamine 2 receptor expression in the nucleus accumbens and medial prefrontal cortex while also enhancing dopamine 2 receptor expression on NK and B cells. Additionally, ethanol was found to reduce desensitization of dopamine 2 receptors in the ventral tegmental area. These results demonstrate that activation of peripheral dopamine 2 receptors can enhance dopamine levels in the nucleus accumbens and that this effect has relevance in understanding the effects of ethanol on dopamine release in the mesolimbic pathway. These results also provide evidence for transient upregulation of dopamine 2 receptors in the brain and on leukocytes suggesting that dopamine 2 receptor levels on leukocytes may be a useful biomarker for central dopamine function.

The distribution of urotensin-II-immunoreactivity (irU-II) was studied in the rat brainstem and spinal cord with the use of an antiserum against the human urotensin II (U-II) peptide. A population of ventral horn neurons in the spinal cord, hypoglossal nucleus, dorsal motor nucleus of the vagus, facial motor nucleus, nucleus ambiguus, abducens nucleus and trigeminal motor nucleus exhibited irU-II of varying intensities. The number of irU-II motor neurons was higher in the lumbar segments as compared to that of cervical, thoracic and sacral segments. Double-labeling the sections with U-II- and choline acetyltransferase (ChAT)-antisera revealed that nearly all irU-II ventral horn and brainstem neurons were ChAT-positive. The result provides the first immunohistochemical evidence of the presence of irU-II in cholinergic motoneurons of the rat spinal cord and brainstem.

Rationale: There is evidence for a common genetic link between alcohol and nicotine dependence. Rodents selectively bred for high alcohol consumption/responsivity are also more likely to self-administer nicotine than controls. Objectives: The experiments examined the response to systemic nicotine, the effects of nicotine within the drug reward pathway, and innate expression of nicotine-related genes in a brain region regulating drug reward/self-administration in multiple lines of rats selectively bred for high and low alcohol consumption. Methods: The experiments examined the effects of systemic administration of nicotine on locomotor activity, the effects of nicotine administered directly into the (posterior ventral tegmental area; pVTA) on dopamine (DA) release in the nucleus accumbens shell (AcbSh), and innate mRNA levels of acetylcholine receptor genes in the pVTA were determined in 6 selectively bred high/low alcohol consuming and Wistar rat lines. Results: The high alcohol-consuming rat lines had greater nicotine-induced locomotor activity compared to low alcohol-consuming rat lines. Microinjections of nicotine into the pVTA resulted in DA release in the AcbSh with the dose response curves for high alcohol-consuming rats shifted leftward and upward. Genetic analysis of the pVTA indicated P rats expressed higher levels of α2 and β4. Conclusion: Selective breeding for high alcohol preference resulted in a genetically divergent behavioral and neurobiological sensitivity to nicotine. The observed behavioral and neurochemical differences between the rat lines would predict an increased likelihood of nicotine reinforcement. The data support the hypothesis of a common genetic basis for drug addiction and identifies potential receptor targets.

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